Antimalarial Activities of Alkyl Cyclohexenone Derivatives Isolated

Article pubs.acs.org/jnp

Antimalarial Activities of Alkyl Cyclohexenone Derivatives Isolated from the Leaves of Poupartia borbonica Allison Ledoux,*,† Alexis St-Gelais,†,§ Ewa Cieckiewicz,† Olivia Jansen,† Annélise Bordignon,† Bertrand Illien,⊥ Nicolas Di Giovanni,∥ Arnaud Marvilliers,⊥ Floriane Hoareau,⊥ Hélène Pendeville,∇ Joel̈ le Quetin-Leclercq,‡ and Michel Frédérich† †

Laboratory of Pharmacognosy, Center for Interdisciplinary Research on Medicines (CIRM), University of Liège, Avenue Hippocrate 15, 4000 Liège, Belgium ‡ Pharmacognosy Research Group, Louvain Drug Research Institute, Université Catholique de Louvain, Avenue E. Mounier, B1 72.03, B-1200 Brussels, Belgium § Laboratoire d’Analyses et de Séparation des Essences Végétales (LASEVE), Université du Québec à Chicoutimi, 555 Boulevard de l’Université, Saguenay, Québec G7H 2B1, Canada ⊥ Laboratoire de Chimie des Substances Naturelles et des Sciences des Aliments (LCSNA), University of Reunion Island, Avenue René Cassin 15, 97744 Saint-Denis, La Réunion France ∥ Laboratoire de Chimie Analytique Organique et Biologique (OBiAChem), University of Liège, Allée de la Chimie 3, Sart-Tilman, 4000 Liège, Belgium ∇ Plateforme Zebrafish Facility and Transgenics, GIGA, University of Liège, Avenue Hippocrate 15, 4000 Liège, Belgium S Supporting Information *

ABSTRACT: Bioactivity-guided fractionation of the ethyl acetate extract of the leaves of Poupartia borbonica led to the isolation of three new alkyl cyclohexenone derivatives 1−3, and named Poupartone A−C. The structures of the new compounds were elucidated by 1D and 2D NMR spectroscopic data analysis and MS, whereas calculated and experimental ECD spectra were used to define the absolute configurations. These compounds were active against 3D7 and W2 Plasmodium falciparum strains with IC50 values between 0.55 and 1.81 μM. In vitro cytotoxicity against WI38 human fibroblasts and the human cervical cancer cell line HeLa (WST-1 assay) showed that these compounds were also cytotoxic, but no hemolytic activity was observed for the extract and pure compounds. An in vivo antimalarial assay was performed on the major cyclohexenone using P. berghei-infected mice at a dose of 15 mg/kg/day ip. The assay revealed growth inhibition of 59.1 and 69.5% at days 5 and 7 postinfection, respectively, although some toxicity was observed. Zebrafish larvae were used as a model to determine the type of toxicity, and the results showed cardiac toxicity. The methanol extract was also studied, and it displayed moderate antiplasmodial properties in vitro. This extract contained the known flavonoids, quercetin, 3′-O-hydroxysulfonylquercetin, quercitrin, and isoquercitrin as well as ellagic acid, which showed high to low activity against the 3D7 P. falciparum strain.

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pharmacophore (18.8%). 3 The endemic plants of the Mascarene Islands appear to be good candidates for study because of their scarcity; most of them are being pharmacologically studied for the first time, increasing the chances for the discovery of new active compounds. Poupartia borbonica J.F. Gmel is a dioecious endemic plant from the Mascarene Islands and belongs to the genus Poupartia in the Anacardiaceae family, as verified in accordance with the International Plant Names Index. The genus is dedicated to the

alaria is caused by Plasmodium, a protozoan parasite transmitted by anopheles mosquitoes, and was responsible for 438 000 deaths worldwide in 2015, according to the last World Malaria report.1 The resistance of parasites to available and affordable medicines has become a widespread problem in exposed countries, making the search for new antimalarial compounds essential. As the immeasurable therapeutic potential of plants is well-established,2 natural products could be an interesting source of new antimalarial drugs. Indeed, according to Newmann and Cragg, more than 60% of the antiparasitic drugs discovered between 1981 and 2014 were unaltered natural products (12.5%), natural product derivatives (31.3%), or synthetic drugs with a natural product © 2017 American Chemical Society and American Society of Pharmacognosy

Received: November 7, 2016 Published: May 30, 2017 1750

DOI: 10.1021/acs.jnatprod.6b01019 J. Nat. Prod. 2017, 80, 1750−1757

Journal of Natural Products

Article

analysis. 3′-O-Hydroxysulfonylquercetin was assigned the formula C15H10O10S from the [M + H]− ion at m/z = 381.5998 in the ESI-HR-TOF-MS data (calcd for C15H10O10S, 381.9995). The presence of an ion at m/z 383.5998 (5.05%) confirmed the presence of a sulfur atom. UV data analysis confirmed the presence of an auxochrome group and a modification of the quercetin B-ring. Similar UV spectra have been described by O’Leary and co-workers.12 The position of the hydroxysulfonyl group was deduced from the chemical shift of H-2′ compared to a quercetin standard. Quercetin is a natural flavonoid found in a wide range of plants and is known to have multiple therapeutic properties, such as anticancer, anti-inflammatory, antiobesity, and antimicrobial activities, as reviewed by D’andrea13 and by Gupta et al.14 Even though data concerning the antiplasmodial activity of quercetin have shown that this flavonol inhibits the growth of different P. falciparum strains,14 its clinical application may be limited because of its low solubility in aqueous media. On the other hand, it has been shown that increasing the solubility of quercetin by preparing aminoalkylated analogues improved its antiplasmodial activity.15 The presence of a sulfate group on quercetin may act similarly by increasing solubility. The antiplasmodial activities of both 3′-O-hydroxysulfonylquercetin and quercetin were evaluated on the P. falciparum 3D7 strain. The IC50 value of quercetin was assessed in this study at 17.54 ± 3.9 μM, in agreement with reported data.16 The 3′-O-hydroxysulfonylquercetin was found to be less active, with an IC50 value of 40.58 ± 8.1 μM. This decrease in activity, despite the higher solubility compared to quercetin, may be due to the steric bulk of the B-ring. In contrast, an aminoalkylated group added to the A-ring of quercetin increased its antiplasmodial activity.15 Isoquercitrin and ellagic acid, whose antiplasmodial activities corresponded to reported data,16,17 were also tested. Their IC50 values were calculated to be 42.34 ± 5.4 μM and 2.6 ± 0.98 μM, respectively. The crude EtOAc extract was found to exhibit the highest activities. Fractionation of the EtOAc extract on an open column led to eight fractions, A to H. Evaluation of the antiplasmodial activity of these fractions showed that fractions E and G were the most active, with IC50 values of 0.38 and 0.72 μg/mL, respectively. Purification of fractions E and G using preparative HPLC afforded three compounds 1−3, which were identified using NMR and LC-MS analysis (Chart 1). Their IR spectra showed a broad OH absorption band at 3383 cm−1 and a strong absorption band of a conjugated carbonyl moiety at 1667 cm−1. The three compounds were not separable by TLC but were separable by reverse-phase HPLC, even though the difference in retention times between 1 and 2 was small. Compound 1 was assigned the formula C23H38O4 from the [M + H]+ ion at m/z = 379.2845 by HR-ESI-TOF-MS analysis (calcd for C23H39O4 = 379.2848). 13C APT NMR and editedHSQC experiments indicated the presence of 1 methyl, 5 methine (including 4 olefinic), 14 methylene, and 3 carbons with no attached protons, including a carbonyl function. Examination of the COSY spectrum showed that most of the methylenes were involved in an alkyl chain bearing one of the two olefinic functionalities and terminated by Me-17′. The other end of this spin system consisted of methylene H-1′, methine H-2′, and methylene H-3′. Both H-1′ and H-5 correlated with the deshielded C-1 and C-6 resonances. The former had a chemical shift that was characteristic of a hemiketal. Additionally, the deshielded C-2′ resonance

botanist, physician, and anatomist François Poupart (1661− 1709), who described many new Poupartia species, including P. borbonica.4 This plant, commonly known in Reunion Island as “Bois blanc rouge” (“red white wood”), is used in traditional medicine to induce a contraceptive effect.5 In the past, it was also used to treat nephritis and boils.6 Indeed, the bark of P. borbonica has an allegedly diuretic effect.7 As far as can be established, the leaves of P. borbonica have not been either phytochemically or pharmacologically studied. Preliminary work (Table 1) demonstrated that several extracts of this plant possess interesting in vitro antiplasmodial activities. This study was designed to determine the compounds responsible for this activity.

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RESULTS AND DISCUSSION Different extraction solvents of dried and pulverized P. borbonica leaves were tested to select the starting point of the bioassay-guided fractionation. The resulting extracts were tested against the P. falciparum 3D7 strain (Table 1). Table 1. Antiplasmodial Activity of the Extracts of P. borbonica Leaves against P. falciparum extraction solvent

yield (% m/m)

EtOAc n-hexane CH2Cl2 MeOH

1.66 1.13 1.05 19.04

IC50 (μg/mL) n = 3 2.43 3.28 3.25 16.23

± ± ± ±

0.5 0.2 0.7 4.3

First, the methanol extract, obtained with a much higher yield, was phytochemically investigated to determine its major compounds. Five known phenolic compounds were isolated: ellagic acid and the four flavonoids, isoquercitrin, quercitrin, 3′O-hydroxysulfonylquercetin, and quercetin, which showed high to low activity against the P. falciparum strain. The structures of these known compounds, except 3′-O-hydroxysulfonylquercetin, were elucidated by comparison of their 1H NMR and UV spectra and HPLC retention behaviors against standard compounds. The major compound of this extract was 3′-O-hydroxysulfonylquercetin, a rare flavonoid inorganic ester. It was isolated by preparative HPLC using a mobile phase containing TFA (0.05%). When the solvent of the fraction was removed under vacuum in a rotary evaporator at a temperature < 30 °C, degradation of the 3′-O-hydroxysulfonylquercetin to quercetin was observed based on the HPLC retention time. The acid of the mobile phase was then changed to HOAc (0.1%) and eventually to formic acid (0.1%). However, this mobile-phase modification did not preclude hydrolysis of the hydroxysulfonyl ester. To avoid this problem, the TFA was removed from the fraction directly by liquid/liquid extraction using Et2O, and the aqueous fraction was then lyophilized. The instability of these types of compounds is the main reason why they are not often isolated. However, sulfate conjugation of flavonoid compounds has been widely observed in many plant families8 to facilitate their storage.9 Hydroxysulfonylquercetin compounds have been reported in the Asteraceae genus in the species Oenanthe crocata, Flaveria bidentis, and Flaveria choloraefolia,8,10 as well as in Cuphea carthagenensis (Lythraceae).11 The structure of 3′-O-hydroxysulfonylquercetin was established by comparison with spectra recorded for a quercetin standard by mass spectrometry, 1H NMR, and UV data 1751



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integrated for two more protons than in compound 1. Thus, the structure of compound 2 was assigned as (2R,3aR,7aR)-2pentadecyl-3a,7a-dihydroxy-2,3,3a,7a- tetrahydrobenzofuran5(4H)-one, and named Poupartone B. Compound 3 has the same molecular formula as 1, given its HR-ESI-TOF-MS ion at m/z = 379.2843. However, while the aliphatic chain was the same as in compound 1, its spectra differed from the two former compounds with respect to the cyclic part of the molecule. A two-proton spin system of the olefinic moiety and a three-proton spin system comprising H-6 and Hz-5 were evident in the 1H NMR spectrum. The H-6 resonance was deshielded, indicating C-6 substitution by a hydroxy group. Both H-5 and H-3 showed HMBC cross-peaks with the C-1 carbonyl. Additional HMBC cross peaks, including H-2/C-4 and H-6/C-4, completed a six-membered ring with the deshielded C-4 carrying the aliphatic chain and an additional hydroxy group. H-1′ exhibited HMBC cross-peaks with both C-4 and the C-2′ carbonyl carbon. A spin system starting from H-3′ constituted the remainder of the aliphatic chain. Thus, compound 3 was found to be a 2′-oxo analogue of 4,6-dihydroxy-4-[(10′Z)-heptadecenyl]cyclohexenone,18 and its structure was defined as (4S,6R)-4,6-dihydroxy-4-[(Z)-2oxoheptadec-10-en-yl]-cyclohexen-2-enone, and named Poupartone C. The key correlations observed in the HMBC and COSY NMR spectra of compounds 1−3 are shown in Figure 1.

Chart 1

suggested that it was bonded to an oxygen. Because only two protons of hydroxy groups were visible in the 1H NMR spectra and neither correlated to C-2′ in the HMBC spectrum, it was deduced that C-1 and C-2′ were linked through an ether bond, with C-1 bearing an additional hydroxy group, in accordance with its chemical shift. The more shielded C-6 was thus connected to both C-1′ and C-5 and to a hydroxy group. This was in accordance with the observed HMBC cross-peaks of both hydroxy protons and C-6. The 1-OH proton also correlated with C-2, whose proton was part of an olefinic spin system that included H-3 as per the COSY spectrum. H-2 further exhibited an HMBC cross-peak with C-4, while H-3 correlated with C-5, suggesting a six-membered ring comprising C-1 through C-6. The remaining HMBC cross-peaks were consistent with the proposed bicyclic structure. TOCSY experiments with increasing mixing times indicated that the acyclic olefinic group was closer to the terminal methyl group than the bicyclic functionality. Its position was inferred from the literature, as all known Anacardiaceae cyclohexenones possess Δ10′(11′) double bonds. This position was confirmed by an intense ion at m/z 145 observed in the GC-EI-MS spectrum of an α,β-bis(methylthio)-derivative of compound 1, obtained by a DMDS derivatization procedure. The (Z) configuration of the Δ10′(11′) olefinic moiety was confirmed by comparison of the chemical shifts of C-9′ and C-12′ with literature values.8 Thus, the structure of compound 1 was defined as (2R,3aR,7aR)-2-[(Z)-pentadec-8-en-1-yl]-3a,7a-dihydroxy2,3,3a,7a-tetrahydrobenzofuran-5(4H)-one, and named Poupartone A. The NMR spectra of compound 2 indicated that it featured the same fused ring structure as 1 with a saturated aliphatic chain, while its HR-ESI-TOF-MS sodium adduct ion at m/z = 403.2812 indicated the presence of two more protons for a formula of C23H40O4 (calcd for [C23H40O4Na]+, 403.2824). The broad multiplets of the aliphatic methylene protons

Figure 1. Key correlations observed in the HMBC ( ( ) NMR spectra of compounds 1−3.

) and COSY

The absolute configurations of compounds 1−3 were established by comparison of their experimental electronic circular dichroism (ECD) and 13C NMR data with those predicted using the DFT/ωB97XD/6-31+G(d,p) method. The modified probability (DP4+) method was used, which differs from the DP4 method by the inclusion of unscaled data and the use of higher levels of theory for the NMR calculation procedure.19 On the basis of geometrical considerations, four possible isomers were conceivable: 1S,6R,2′R/1R,6S,2′S, 1S,6R,2′S/1R,6S,2′R, 1S,6S,2′R/1R,6R,2′S, and 1S,6S,2′S/ 1R,6R,2R (Figure 2). The comparison of experimental and calculated NMR spectra allowed the determination of the relative configuration of 1 and 2 as (1S,6S,2′S/1R,6R,2′R) with a DP4+ probability of 100%. The (1R,6R,2′R) absolute configuration was then assigned by comparing the experimental and calculated ECD 1752



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Using the method described by Deharo and Ginsburg,20 the cyclohexenones 1−3 explain almost 50% of the activity of the EtOAc crude extract against P. falciparum (compound 1: 21.5%; compound 2: 21.4%; compound 3: 2.9%). As compound 1 was the most abundant, an in vivo antimalarial assay was performed using P. berghei-infected mice. Compound 1 showed an inhibition of 59.1 and 69.5% of parasitic growth at days 5 and 7 postinfection, respectively (Figure 3). Compound 1 significantly reduced parasitemia compared to the negative control. However, it was not devoid of toxicity, and despite the reduction of parasitemia, mice started to die on day 7. Zebrafish larvae were used as a model to observe the toxicity of compound 1. The embryonic development of zebrafish is rapid, and the optical transparency of the embryos is convenient for investigating the causes of lethality.25 It is also possible to observe developmental effects of a drug. Even if zebrafish cannot replace rodent models, they are useful for rapidly determining the toxicity of a drug in the early stages of an investigation. Embryos were dechorionated because the chorion has a protective function and isolates the embryo physically and chemically from external environmental conditions.26 In the first step of analysis, logarithmic concentration series were used for range-finding. During the period of 24 h postfertilization (hpf) to 72 hpf, it was observed that all embryos died within 1 day after introduction of 10 μg/ mL of compound 1. The second part of the test enabled more detailed observations of toxicity at a range of specific drug levels. After the first treatment doses, all embryos died at doses of 10, 5, or 3 μg/mL. At these concentrations, the embryos disintegrated, suggesting high contact toxicity. Neither 2.5 nor 2 μg/mL doses induced disintegration, but a few (5%) of the embryos developed bradycardia. No toxicity was observed at 1 μg/mL. Cumulative mortality was observed at 48 and 72 hpf, after two and three treatment doses, respectively. The 2.5 μg/ mL concentration dose was lethal for all embryos at 48 hpf. At 2 μg/mL, 60% of the embryos died at 48 hpf, while the survivors developed a large decrease in pericardial function and body axis deformation and died at 72 hpf after the third dose. In contrast, at 72 hpf, no significant mortality or morphological modifications were observed in embryos treated with 1 μg/mL. The toxicity results from using a zebrafish embryo model may be an indication of the toxicity observed in mice. Indeed, although their cardiac morphology is simpler than the mammalian one, many features of the zebrafish heart are similar to those of mice.27 In conclusion, the bioguided fractionation of an extract of the leaves of P. borbonica led to the isolation of three new alkyl cyclohexenone derivatives (1−3) that could explain, at least in part, the antiplasmodial activity of the EtOAc crude extract. These compounds showed high in vitro antiplasmodial activities, without any hemolysis, on the chloroquine-sensitive P. falciparum strain as well as on the resistant strain and displayed some toxicity. The in vivo assays performed on mice confirmed the high antimalarial activity of compound 1, even if toxicity was recorded from day 7. Regarding the toxicity test on the zebrafish embryo model, compound 1 seems to be responsible for heart failure. The mechanisms of action of these high potential antiplasmodial cyclohexenones, as well as elucidation of their structure−activity relationships, still need to be investigated. Ellagic acid, which is known to have high antiplasmodial activity,17,29 seems to be the compound that explains the major part of the activity of the methanol crude extract, even if other compounds may act in synergy or have an

Figure 2. Diagram of the most stable configurations of the four isomers of compound 1.

spectra at the TDA/RI-B2GP-PLYP/def2-TZVPP/SMD level in MeOH (Supporting Information). The comparison of the experimental and calculated NMR spectra of compound 3 allowed the definition of the relative configuration as (4R,6S/4S,6R) with a DP4+ probability of 100%. The (4S,6R) absolute configuration was then assigned by comparing the experimental and calculated ECD spectra at the TDA/RI-B2GP-PLYP/def2-TZVPP/SMD level in MeOH. The compounds from the EtOAc extract were tested in vitro on the parasitic protozoa P. falciparum, and all showed high levels of activity against both chloroquine-sensitive strain W2 and chloroquine-resistant strain 3D7 (Table 3) with IC50 < 2 mM. This class of compounds was previously observed in the Anacardiaceae family in Tapirira guianensis and showed antiprotozoal and antibacterial activities. 21 One of the compounds, described by Roumy et al. as 1,2′,4,5-tetrahydroxy-1-[10′Z-heptadecenyl]-2-cyclohexene, had a much lower activity level (86.4 ± 2.6 μg/mL on P. falciparum).21 Thus, it seems there is a positive effect on activity by the presence of a C-1 carbonyl and of an oxidation on C-2′ for compound 3. Similar observations can also be established for compounds 1− 2 possessing Δ2(3) double bonds, as the compound devoid of this double bond, 1,3,4,6-tetrahydroxy-1,2′-epoxy-6-[10′Zheptadecenyl]cyclohexane, is less active, with an IC50 on Plasmodium falciparum F32 strain of 101.3 ± 3.8 μM.21 Other cyclohexenone derivatives were also described in Tapirira obtusa,22 as well as in Pistacia vera.23 The cytotoxic activities of these types of compound were highlighted in Tapirira guianensis.18 A Chinese patent (CN 1733748 A, Feb 15, 2006) was filed for a compound that has a structure similar to that of compound 1 but with a longer side chain and a different configuration of the bicyclic system. It was obtained from extracts of Choerospondias axillaris, a plant from the Anacardiaceae, and possesses cell cycle inhibitory and apoptosis inductor effects allowing its use as an antitumor and anticancer agent.24 This finding was a factor in the decision to analyze the cytotoxicity of 1−3. These results show that compounds 1−3 are the bioactive compounds responsible for the activity of the EtOAc extract of the leaves of P. borbonica but also for the cytotoxicity which is similar to related compounds.21 Compound 1 has the most promising activity but is also the most cytotoxic, with an SI of 1.76 on the P. falciparum 3D7 strain and WI38 cells. However, the antiplasmodial activity was not due to hemolysis, because neither the extract nor the isolated compounds induced red blood cell lysis at the highest concentration tested. 1753


1754

44.3, CH2

77.6, CH

37.0, CH2 26.6, CH2 30.1−30.7, CH2

28.03, CH2

131.03, CH 130.98, CH

27.98, CH2 30.65, 29.9, CH2 32.7, CH2 23.6, CH2 14.6, CH3

2′

3′ 4′ 5′-8′

9′

10′ 11′

12′ 13′-14′

a

1

1.25−1.35 (m) 1.25−1.35 (br. m) 0.88 (t, J = 7.3)

2.02 (m) 1.25−1.35 (m)

5.35 (m) 5.35 (m)

2.02 (m)

4.65 (s) 3.72 (s) 2.14 (dd, J = 13.2, 6.7); 1.62 (dd, J = 13.2, 8.5) 4.28 (ddd, J = 13.2, 8.5, 6.7) 1.50 (m); 1.40 (m) 1.25−1.35 (br. m) 1.25−1.35 (br. m)

2.70 (dd, J = 15.9, 0.7); 2.57 (d, J = 15.9)

6.65 (d, J = 10.2) 5.83 (dd, J = 10.2, 0.7)

H (J in Hz)

1

17′ 17′

6′, 7′, 8′, 10′, 11′ 7′, 8′, 9′, 11′ 7′, 8′, 9′, 10′, 12′ 10′, 11′ 12′, 15′, 16′

1′, 2′ 3′ 4′, 10′, 11′

1′, 3′

6, 5

1, 2, 5, 1′

3, 5, 6, 1′ 1 5 2, 5 1′, 3, 6

HMBC C→H

C, type

32.7 CH2 23.5 CH2 14.5, CH3

36.9, CH2 26.5, CH2 30.44, 30.43 (2×), 30.41 (2×), 30.33 (2×), 30.32 (2×), 30.1, CH2

77.4, CH

44.2, CH2

79.7, C

100.4, C 147.3, CH 127.3, CH 198.2, C 48.2, CH2

13

H (J in Hz)

1

1.24−1.34 (br. m) 1.24−1.34 (br. m) 0.88 (t, J = 7.1)

1.24−1.34 (br. m) 1.24−1.34 (br. m)

1.24−1.34 (br. m) 1.24−1.34 (br. m)

1.24−1.34 (br. m)

not visible not visible 2.15 (dd, J = 13.1, 6.7); 1.62 (dd, J = 13.1, 8.4) 4.28 (ddd, J = 13.1, 8.4, 6.7) 1.51 (m); 1.39 (m) 1.24−1.34 (br. m) 1.24−1.34 (br. m)

2.70 (dd, J = 16.1, 0.4); 2.57 (d, J = 16.1)

6.65 (d, J = 10.1) 5.83 (dd, J = 10.1, 0.4)

2

17′

6′, 7′, 8′, 10′, 11′ 7′, 8′, 9′, 11′ 7′, 8′, 9′, 10′, 12′ 10′, 11′ 12′, 15′, 16′

1′ 2′, 5′, 6′, 7′ 4′

2, 5, 1′, 3′

5

3, 5, 1′ 1 5 2, 5 3, 1′

HMBC C→H

C, type

32.8, CH2 23.1, CH2 14.3, CH3

27.6, CH2

130.84, CH 130.79, CH

45.6, CH2 24.1, CH2 30.5, 30.23, 30.21, 30.17, 30.0, 29.8, CH2 27.8, CH2

210.2, C

48.7, CH2

65.9, CH

200.7, C 126.3, CH 154.2, CH 75.8, C 44.7, CH2

13

In CD3CN/methanol-d4 1:1, referenced to CD3CN residual peak bIn methanol-d4, referenced to TMS cIn CD3CN, referenced to solvent residual peak

15′ 16′ 17′

79.9, C

6 1-OH 6-OH 1′

C, type

100.5, C 147.4, CH 127.5, CH 198.3, C 48.4, CH2

1 2 3 4 5

position

13

Table 2. NMR Data of Compounds 1−3 (δ in ppm, Multiplicity (J in Hz) and HMBC Correlations) 3

1.23−1.34 (br. m) 1.23−1.34 (br. m) 0.89 (t, J = 7.4)

2.02 (m) 1.23−1.34 (br. m)

5.35 (m) 5.35 (m)

2.02 (m)

2.47 (d, J = 8.5) 1.47 (m) 1.23−1.34 (br. m)

2.64 (d, J = 14.7); 2.59 (d, J = 14.7)

2.50 (ddd, J = 12.9, 4.9, 2.0); 1.80 (dd, J = 12.9, 9.1) 4.51 (ddt, J = 9.1, 4.9, 2.0)

5.95 (dd, J = 10.2, 2.1) 6.90 (dt, J = 10.2, 2.0)

H (J in Hz)

1

17′ 17′

10′, 11′, 13′ 12′, 15′, 16′

11′ 10′

10′, 11′, 12′

1′, 4′, 5′, 6′ 3′ 10′, 11′

1′, 3′

6, 3′

2, 5

3

3, 5, 1′ 3, 6 5, 1

HMBC C→H

Journal of Natural Products Article



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Table 3. Antiplasmodial and Cytotoxic Activities of Compounds 1−3 and the EtOAc Extract

samples 1 2 3 EtOAc extract artemisinin camptothecin

P. falciparum IC50 (μg/mL)

cytotoxicity IC50 (μg/mL)

IC50 (μM) (n = 3)

IC50 (μM) (n = 2)

W2

3D7

0.21 ± 0.010 0.55 ± 0.003 0.37 ± 0.050 0.97 ± 0.130 0.48 ± 0.040 1.27 ± 0.105 1.78 ± 0.390 0.010 ± 0.002 0.035 ± 0.007 NT

0.34 ± 0.040 0.90 ± 0.015 0.69 ± 0.200 1.81 ± 0.520 0.43 ± 0.160 1.13 ± 0.420 2.43 ± 0.500 0.004 ± 0.001 0.014 ± 0.004 NT

HeLa 0.80 2.10 1.44 3.77 1.31 3.45 3.37

± 0.120 ± 0.316 ± 0.340 ± 0.890 ± 0.430 ± 1.130 ± 0.350 NT

0.080 ± 0.015 0.230 ± 0.043

0.60 1.58 1.04 2.73 1.01 2.66 3.23

± 0.380 ± 1.000 ± 0.500 ± 1.300 ± 0.820 ± 2.160 ± 0.350 NT

0.030 ± 0.011 0.086 ± 0.032

(SI) WI38/W2

WI38/3D7

hemolysis (%)

2.86

1.76

Antimalarial Activities of Alkyl Cyclohexenone Derivatives Isolated

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